Members of the Order Spirochaetales include organisms responsible for diseases associated with significant morbidity and mortality in humans and other animals. The spirochetes are helically shaped, motile bacteria that stain as Gram negative, and include the genera Borrelia, Brachyspira, Cristipira, Leptonema, Leptospira, Serpulina, Spirochaeta, Treponema, Pillotina, Diplocalyx, Hollandina, and Clevelandina. Of these genera, the Borrelia, Leptospira, and Treponema are responsible for the majority of human disease.
The Spirochetes
The genus Borrelia includes numerous species, the most clinically significant of which is B. burgdorferi, the etiologic agent of Lyme disease. Other Borrelia are historically significant as causative agents of epidemics, including B. recurrentis (synonyms include B. obermeyeri and B. novyi), B. duttoni, B. hermisii, and various others. For example, during the first half of the 1900s, more than 50 million people contracted louse-borne relapsing fever, with epidemics occurring throughout Europe, Africa, Asia, and South America (Schwan et al., Borrelia, in Murray (ed.), Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C. [1995], pages 626-635). Since 1959, more than 840 cases, including at least 6 deaths have been recorded from Jordan, Rwanda and Iran (See, Schwan et al., supra).
The genus Leptospira includes pathogenic as well as non-pathogenic serovars, although the pathogenicity is not a criterion for species differentiation. Nonetheless, the pathogenic serovars have been traditionally included within the species L. interrogans and the free-living non-pathogenic serovars have been included within the species L. biflexa. Over 210 serovars of L. interrogans and 63 serovars of L. biflexa have been officially described (Kaufmann and Weyant, Leptospiraceae, in Murray (ed.), Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C. [1995], pages 621-625). Leptospirosis is a zoonotic disease, with reservoirs in wild, domestic and feral animals. The disease can be very serious in humans and other animals, most notably pinnipeds. In humans, it is characterized by a biphasic illness in which about 10% of patients develop icteric leptospirosis. Icteric leptospirosis can be clinically severe, with a mortality rate of approximately 10% (See, Kaufmann and Weyant, supra).
The genus Treponema includes four human pathogens and at least six species that are not human pathogens. The most clinically significant species is T. pallidum, which includes three subspecies. T. pallidum subsp. pallidum is the etiologic agent of venereal syphilis, while T. pallidum subsp. pertenue is the etiologic agent of yaws (frambesia, pian), and T. pallidum subsp. endemicum is the etiologic agent of endemic syphilis (bejel, dichuchwa). T. carateum, the etiologic agent of pinta (carate, cute) represents the fourth pathogenic organism within this genus. Despite the availability of effective therapy, venereal syphilis remains an important sexually transmitted disease with a worldwide distribution. For example, in the United States, 112,581 cases were reported in 1992, which included 33,973 cases of primary and secondary syphilis and 3,850 cases of congenital syphilis (Norris and Larsen, Treponema and Other Host-Associated Spirochetes Murray (ed.), Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C. [1995], pages 636-651). Although venereal syphilis is of greatest concern in the United States, the other forms of treponemal disease are significant in terms of morbidity and mortality worldwide. Endemic syphilis is restricted to the desert and temperate regions of North Africa and the Middle East, while yaws occurs most frequently in the tropical and desert regions of Africa, South America, and Indonesia, and pinta is primarily observed in tropical areas of Central and South America (Norris and Larsen, supra). Yaws, endemic syphilis and pinta were endemic in certain areas prior to the establishment of eradication programs by the World Health Organization. In the 1950s, it was estimated that 200 million people were exposed to these diseases (Norris and Larsen, supra).
Cultivation and Identification of Spirochetes
In general, the spirochetes are difficult to culture and some have a requirement for in vivo cultivation methods. Indeed, Lyme disease spirochetes are difficult to detect in human patients. However, Borrelia can be cultivated either in their arthropod vectors or in a large variety of vertebrate hosts, although cultivation in embryonated chicken eggs is also possible. In vitro cultivation of Borrelia is most often done using Barbour-Stoenner-Kelly II (BSK II) medium, pH 7, with incubation in a microaerophilic environment at 30° to 37° C. The culture medium is monitored for spirochetes by dark field microscopy for 4 to 6 weeks. Traditionally, cultivation is conducted in the presence of gelatin and rabbit serum, as it has been reported that many months of incubation may be required to successfully grow these organisms in the absence of these components (See, Schwan et al., supra). An additional consideration is that continuous serial passages, even over a short period may effect many biological changes in the organisms, altering the phenotypic and genotypic characteristics.
Historically, identification of the Borrelia heavily depended upon the geographic distribution and natural arthropod vectors. The development of molecular diagnostic procedures has greatly enhanced the ability to identify these organisms, although direct identification of B. burgdorferi in clinical material by polymerase chain reaction (PCR) has proven unreliable, except in cases where synovial fluid is used (See, Schwann et al., supra). Serologic confirmation of borrelioses is often attempted using methods such as immunofluorescence (IFA) and enzyme immunoassays (ELISA or EIA), and Western blots. In addition, molecular identification methods have been developed.
Disease caused by leptospires is often presumptively diagnosed based on direct detection of organisms in a sample. These methods require skill and experience in order to correctly differentiate organisms from artifacts in the samples. In vitro methods have also been developed to cultivate leptospires. However, their fastidious nature makes this a complicated and time-consuming undertaking. Most often, semisolid medium such as Fletcher's, Ellinghausen's, or polysorbate 80 is used. Traditionally, leptospiral cultures are maintained at room temperature, and cultures are observed once a week, for at least five weeks, using dark field microscopy. Before reporting a culture as “negative,” the culture is examined twice a month for four months. Once grown in culture, leptospires can be identified to serogroup by the microscopic agglutination test (MAT). Other serologic methods have also been developed (e.g., indirect hemagglutination, slide agglutination, and ELISA), although none of these alternative methods appear to have the necessary sensitivity and specificity for clinical diagnostics (See, Kaufmann and Weyant).
The Treponema present even greater challenges to the microbiologist, as the T. pallidum subspecies and T. carateum are obligate human parasites, with no known non-human animal or environmental reservoirs. Thus, diagnosis of such diseases as syphilis is based on direct microscopic examination of material collected from lesions, non-treponemal tests (for screening), and treponemal tests (for confirmation). The criteria for syphilis diagnosis are divided into three categories, namely definitive, presumptive, and suggestive. The Treponemes are reported as noncultivatable in vitro. In the United States, the routine testing scheme is direct microscopic examination of lesion exudates, followed by a non-treponemal test, which is then confirmed with a treponemal test, if positive (See, Norris and Larsen). The diagnosis of other treponemal diseases is even more cumbersome, as no laboratory methods have been developed to distinguish the other pathogenic treponematoses from each other or from syphilis. Indeed, the standard serologic tests for syphilis are uniformly reactive with yaws, pinta, and endemic syphilis (See, Norris and Larsen). Thus, diagnosis of these diseases can be problematic.
Treatment of Spirochetal Diseases
For borrelial infections, there appears to be general agreement that antibiotics are preferable to the arsenical compounds traditionally used to treat these diseases. For Lyme disease, treatment regimens depend upon the nature and severity of the clinical manifestations. Very few prospective randomized therapeutic trials have been conducted, and MICs (minimum inhibitory concentrations) and MBCs (minimum bactericidal concentrations) are inconsistently reported in the literature.
For leptospires, standardized procedures have yet to be developed for antimicrobial susceptibility testing (See, Kaufmann and Weyant). However, in vitro testing has demonstrated strain variability in the susceptibility of the organisms to penicillin and tetracyclines. Indeed, additional methods need to be developed before in vitro testing of leptospires can be recommended for selection of treatment regimens. Furthermore, the limited availability of laboratories with the requisite capabilities for leptospiral disease diagnostics remains a problem worldwide.
In the United States, the Centers for Disease Control (CDC) publish recommended guidelines for the treatment of syphilis. Treatment of syphilis is often empirical, as antimicrobial testing is not straightforward (i.e., due to the lack of a method for continuous culture of T. pallidum). Various approaches been developed to determine the susceptibilities of representative strains (e.g., Nichols strain) to antimicrobial agents based on criteria such as the in vitro loss of mobility or infectivity, treatment of experimental animal infections, human trials, and examination of non-pathogenic cultivable treponemes.